The use of Trichomonas vaginalis purine nucleoside phosphorylase to activate fludarabine in the treatment of solid tumors.

Treatment with fludarabine phosphate (9-ß-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

Intratumoral generation of 2-fluoroadenine to treat solid malignancies of the head and neck.

This report describes treatment of locoregional head and neck squamous cell carcinoma (HNSCC) by an innovative, experimental strategy involving generation of a robust anti-cancer agent (2-fluoroadenine [F-Ade]) following transduction by Escherichia coli purine nucleoside phosphorylase (PNP) in a small number of tumor cells. F-Ade works by a unique mechanism of action (ablation of RNA and protein synthesis) and confers tumor regressions of otherwise refractory HNSCC in human subjects. Clinical studies have now advanced to a pivotal (registration-directed) trial involving locoregional HNSCC, with plans to begin subject enrollment late in 2018. The present review is the first to summarize use of PNP in the context of HNSCC, and provides background regarding this emerging anti-cancer approach.

Alterations of global histone H3K9 and H3K27 methylation levels in bladder cancer.

BACKGROUND: Epigenetic alterations, including histone modifications, play an important role during carcinogenesis. This study was designed to systematically investigate histone H3K9 and H3K27 methylation levels in bladder cancer (BCa) tissue. METHODS: A tissue microarray with urothelial BCa (150 non-muscle-invasive BCa, NMIBC; 121 muscle-invasive BCa, MIBC; 31 metastatic BCa, MET) and normal urothelium (29, CTRL) specimen was used to determine the global levels of H3K9 and H3K27 mono-, di- and tri-methylation. RESULTS: Global levels of H3K9 and H3K27 methylation were significantly higher in CTRL than in BCa, and levels in NMIBC were higher compared to MIBC. Histone methylation levels of MET resembled MIBC. We observed furthermore a correlation of histone methylation levels with pT stage (H3K9me1, H3K9me2, H3K9me3, H3K27me1, H3K27me3) and grade (H3K9me2, H3K9me3, H3K27me1) in NMIBC. H3K9me1, H3K9me3, H3K27me1 and H3K27me3 levels were also correlated with pT stage in MIBC. Histone modifications were not associated with recurrence-free or cancer-specific survival. CONCLUSIONS: Global histone H3K9 and H3K27 methylation levels are altered in BCa.

Identification of prostaglandin receptors in human ureters.

BACKGROUND: Prostaglandins play an important role in ureteral obstruction, but the detailed expression profiles of the prostaglandin receptors (PTGER1, PTGER2, PTGER3, PTGER4, PTGFR) remain unknown in the different parts of the human ureter. METHODS: The expression pattern of PTGER1, PTGER2, PTGER3, PTGER4 and PTGFR was determined in human distal, mid and proximal ureter and renal pelvis samples using immunohistochemistry (protein levels) and quantitative real-time PCR (mRNA). RESULTS: PTGER1 was highly expressed in most samples irrespective of the ureteral localization; however, urothelial cells had higher levels of PTGER1 than smooth muscle cells. PTGFR was also moderately to strongly expressed in urothelial and smooth muscle cells. In comparison, PTGER2-4 expression was mostly unexpressed or weakly expressed in urothelial and smooth cells in all regions. CONCLUSIONS: Our data indicate high levels of PTGER1 in ureters.

Alterations of global histone H4K20 methylation during prostate carcinogenesis.

BACKGROUND: Global histone modifications have been implicated in the progression of various tumour entities. Our study was designed to assess global methylation levels of histone 4 lysine 20 (H4K20me1-3) at different stages of prostate cancer (PCA) carcinogenesis. METHODS: Global H4K20 methylation levels were evaluated using a tissue microarray in patients with clinically localized PCA (n = 113), non-malignant prostate disease (n = 27), metastatic hormone-naive PCA (mPCA, n = 30) and castration-resistant PCA (CRPC, n = 34). Immunohistochemistry was performed to assess global levels of H4K20 methylation levels. RESULTS: Similar proportions of the normal, PCA, and mPCA prostate tissues showed strong H4K20me3 staining. CRPC tissue analysis showed the weakest immunostaining levels of H4K20me1 and H4K20me2, compared to other prostate tissues. H4K20me2 methylation levels indicated significant differences in examined tissues except for normal prostate versus PCA tissue. H4K20me1 differentiates CRPC from other prostate tissues. H4K20me1 was significantly correlated with lymph node metastases, and H4K20me2 showed a significant correlation with the Gleason score. However, H4K20 methylation levels failed to predict PSA recurrence after radical prostatectomy. CONCLUSIONS: H4K20 methylation levels constitute valuable markers for the dynamic process of prostate cancer carcinogenesis.

Pathological outcomes of men eligible for active surveillance after undergoing radical prostatectomy: are results predictable?

INTRODUCTION: To analyze pathological results in patients with prostate cancer eligible for active surveillance (AS) after radical prostatectomy and available prediction systems. METHODS: A retrospective analysis was performed of 612 patients who underwent radical prostatectomy during a 14-year period. Subsequently, we selected those patients who would have been eligible for AS according to 2 different published criteria. Group AS-A matched the following criteria: ≤T2a; Gleason Score ≤6; and prostate-specific antigen <10 ng/mL, while group AS-B applied to different criteria: ≤T2a; Gleason Score <7; and prostate-specific antigen ≤15 ng/mL. Pathological outcomes were compared with results of the 2001 Partin tables. RESULTS: Altogether, 125 (20.4%) patients were included in group AS-A and 159 (25.9%) in group AS-B. We detected 32 cases of >pT2c (25.6%) for group AS-A and 47 cases (29.6%) for AS-B, respectively. Gleason score upgrading was recorded in 34.4% (AS-A) and 38.3% (AS-B). Results of the Partin tables showed good discrimination among patients at risk for positive lymph nodes but limited discrimination for organ-confined disease, seminal vesicle. CONCLUSIONS: Overall >25% of patients eligible for AS showed either upstaging or Gleason score upgrading, which could not be measured with the examined predictive tools. Patients should be informed about the risks of inaccurate preoperative diagnostic.

Tyrosine kinase expression profile in clear cell renal cell carcinoma.

PURPOSE: To profile different tyrosine kinase (TK) expression patterns in clear cell renal carcinoma (ccRCC). METHODS: We analysed mRNA expression levels of 89 receptor and non-receptor TK in corresponding cancer and normal renal tissue from 5 patients with ccRCC using the TaqMan Low-Density Array technology. In order to confirm aberrant TK expressions, a subsequent analysis of 25 ccRCC and corresponding normal renal tissues was performed, applying quantitative real-time PCR. To confirm mRNA expression levels on protein level, we studied ERBB4 and HCK using immunohistochemistry. RESULTS: A total of 12 TK were significantly upregulated in ccRCC (ABL2, FLT1, BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK, PTPRC, FYN and CSK), coherently 7 TK demonstrated a down-regulation (ERBB4, PDGFRA, NRTK3, SYK, ERBB2, FGFR3 and PTK7). These findings were validated by the utilization of RT-PCR for ABL2, FLT1 BTK, HCK, JAK3, CSF1R, MET, JAK1, MATK and vice versa for ERBB4 and PDGFRA. Immunohistochemistry revealed ERBB4 expression to be significantly lower in ccRCC in comparison to papillary RCC, chromophobe RCC, renal oncocytoma and normal renal tissue (P < 0.001). HCK protein expression was reduced in ccRCC in contrast to papillary RCC (P < 0.001) or oncocytoma (P = 0.023), but similar to chromphobe RCC (P = 0.470), sarcomatoid RCC (P = 0.754) and normal renal tissue (P = 0.083). Neither ERBB4 nor HCK were correlated (P > 0.05) with clinical-pathological parameters. CONCLUSION: TK constitute valuable targets for pharmaceutical anti-cancer therapy. ERBB4 and HCK depict significantly lower expression levels in renal cancer tissues.